Autoimmune diseases are characterized by an abnormal immune response (involving either immune system cells or antibodies) directed against normal autologous (self) tissues. Autoimmune diseases afflict huge numbers of individuals throughout the world.
A normal immune system has the capacity to identify and destroy a large variety of foreign invader organisms such as bacteria and viruses. Remarkably, a normal immune system can readily distinguish foreign substances from self, and thereby is able to react vigorously against potentially pathogenic entities from the environment without harming the host's own cells.
The immune system's non-reactivity to self is termed immunological tolerance. In pathological situations, immunological tolerance to a wide variety of self substances is broken, resulting in an autoimmune response to self. If of an appropriate nature and of sufficient severity and duration, the anti-self response will result in an autoimmune disease. In certain autoimmune diseases, specific elements of the immune system predominate in mediating the pathogenic process, while in other autoimmune diseases, all of the components of the immune system cooperate to produce disease. Antibodies are considered to play the major causal roles in diseases such as systemic lupus erythematosus, myasthenia gravis and Graves' disease, while cellular immune mechanisms are believed to be those primarily involved in multiple sclerosis (MS) and insulin dependent diabetes (IDD).
Whereas susceptibility to autoimmune diseases may be inherited through the defective actions of multiple genes, indirect evidence exists to suggest that an interaction with a foreign substance from the environment may also be necessary to induce the pathogenic process that results in disease. One explanation for this is that immunization with the foreign inductive chemical induces a cross-reactive response to self through molecular mimicry or chemical similarity. However, once the autoimmune process has been initiated, other secondary immunizing events involving other self antigens typically occur through the release of intracellular constituents in forms not normally encountered by the immune system. Targeted organs thus become damaged through the combination of all of these events, which will lead to the appearance of a clinically recognized disorder only when the disease process has progressed to result in the ablation of large numbers of tissue cells so targeted.
A number of strategies have been used or proposed to suppress autoimmune diseases, most notably drugs, such as cyclophosphamide, cyclosporin A, methotrexate, and Imuran (azathioprine). Steroid compounds, such as prednisone and methylprednisolone, are also employed in many instances. These drugs have limited long term efficacy against both cell- and antibody-mediated autoimmune diseases. Use of such drugs is limited by virtue of their toxic side effects which include "global" immunosuppression. Prolonged treatment with these drugs inhibits the normal protective immune response to pathogenic microorganisms, thereby increasing the risk of infections. A further drawback is that immune-mediated elimination of aberrant cells is impaired and there is, thus, an increased risk that malignancies will develop in patients receiving prolonged global immunosuppression.
The self substances, or autoantigens, which are the targets of autoimmune responses are most often protein products unique to the targeted cells (e.g., hormones such as insulin in IDD); particular enzymes unique to the specialized function of targeted cells (e.g., glutamic acid decarboxylase or GAD in IDD, or 21 hydroxylase in Addison's disease); specialized cell specific receptor molecules (e.g. the thyroid stimulating hormone or TSH receptor in Graves' disease or acetylcholine receptors in the neuromuscular junctions in myasthenia gravis); and/or structural constituents of the targeted cells or tissues (e.g., beta cell sialo-glycoconjugate in IDD). Prior to the current invention, immunization with autoantigens has been used as a means to induce autoimmune disease in experimental animals. For example, the administration of myelin basic protein (MBP) has been used as a means to induce EAE (a model for MS) in mice.
The methods and compositions of the subject invention are specifically exemplified with regard to the prevention of IDD. However, as discussed more fully herein, the principles discovered by applicants for preventing diabetes are broadly applicable to a variety of known autoimmune conditions as well as to autoimmune conditions which may be identified in the future. Known autoimmune disorders include diabetes, multiple sclerosis, autoimmune uveitis, rheumatoid arthritis, Addison's disease, thyroiditis, atrophic gastritis, myasthenia gravis, idiopathic thrombocytopenic purpura, hemolytic anemia, systemic lupus erythematosus, primary billary cirrhosis, Wegener's granulomatosis, polyarteritis nodosa, and inflammatory bowel disease. Because the subject invention is specifically exemplified with regard to diabetes, a detailed background of diabetes is provided below.
Insulin dependent diabetes. Diabetes mellitus comprises a group of diseases that result in elevations of blood glucose levels because of relative to absolute deficiencies in the pancreatic hormone, insulin. Insulin is secreted into the blood when food is ingested and has its major actions in directing the absorbed nutrients into body stores. Diabetes is a major public health problem affecting at least 5 and as many as 10 million Americans. The prevalence of the most severe form of IDD is 1 in 300 in the United States. In 1991, the direct health care costs attributable to diabetes care in the United States exceeded $20 billion, with as much as twice this figure in additional indirect costs, such as for loss of productivity (Bransome and Edwin 1992! Diabetes Care 15:1-5).
Chronic elevation of blood glucose levels is the most obvious metabolic effect in diabetes and is associated with progressive damage to blood vessels. This leads to heart attacks, strokes, blindness, peripheral nerve dysfunction, and kidney failure. The frequency and severity of diabetes related complications are greatest in the insulin dependent form of the disease, in which an immunological destruction of the pancreatic insulin-secreting beta cells occurs. The high rate of irreversible complications in IDD occurs despite the availability of insulin replacement through injections given 1-4 times daily.
Insulin, as well as other pancreatic hormones, are well known and characterized. See, for example, Steiner et al. (1989) "Chemistry and Biosynthesis of Pancreatic Protein Hormones," In Endocrinology, DeGroot et al., Eds., W. B. Saunders Company, pp. 1263-1289. As described in Steiner et al., the amino acid sequence of insulin is highly conserved across a number of species, including human, monkey, porcine, and bovine. Although insulin is well known for its association with diabetes, there are a number of other proteins which are also associated with the pancreas or diabetes. These other proteins include glucagon and glutamic acid decarboxylase (GAD).
Through the research efforts of ourselves and others, IDD has proved itself to be predictable both in unaffected relatives of patients with IDD, as well as in persons from the general population. A predisposition to develop clinical diabetes can be determined through several different tests. For example, genetic susceptibility to diabetes has become increasingly definable through the use of molecular biological means usually from DNA samples obtained from peripheral blood. One major gene involved in the inherited susceptibility to IDD is that located at the HLA-DQ locus. It is currently possible to identify risks varying from essentially none to those as high as 70 fold above those without the genetic genotype. In families, a genetic risk as high as 1 in 4 can be estimated for unaffected siblings just through identification of HLA haplotypes shared with the affected proband.
Persons who have just developed IDD or are in process of developing IDD have a number of disease specific autoantibodies in their blood. Such autoantibodies include those to islet cell antigens (ICA), to beta cell specific proteins of 64 kDa (now believed to be the lower molecular isoform of glutamic acid decarboxylase GAD.sub.65 !) to native insulin and proinsulin, and to a number of more minor determinants such as carboxypeptidase-H and heat shock proteins belonging to the hsp-60 family.
Insulin autoantibodies (IAA) are observed in untreated, newly diagnosed IDD patients (Palmer et al. 1983! Science 222:1337-1339) as well as in unaffected relatives of diabetic probands. Whereas autoimmunity to insulin could directly cause .beta.-cell damage, interfere with the action of endogenous insulin, or have both effects, some investigators suggested that IAA reflect the rate of islet cell destruction and thus act merely as reporters of aggressive islet directed autoimmunity (Ziegler et al. 1989! Diabetes 38:1320-1325; Vardi 1988! Diabetes Care 11:736-739).
The non-obese diabetic (NOD) mouse is a useful animal model for human IDD. Analysis of the NOD mouse provides important insights into the sequence of pathogenic events, which leads to an understanding of the nature of the target islet cell autoantigens involved in the autoimmunological process. Previous studies from our laboratory have demonstrated that an extended prophylactic course of daily, subcutaneous injections of porcine insulin protected NOD mice from both hyperglycemia and islet infiltration by mononuclear leukocytes (insulitis) (Atkinson et al. 1990! Diabetes 39:933-937). Such treatment may relieve the pancreatic .beta.-cells of metabolic demands on them and thus induce a state of ".beta.-cell rest." This quiescent state may be associated with diminished expression of many islet factors, including those that may serve as potential autoantigens at the cell surface (Aaen et al. 1990! Diabetes 39:697-701; Kampe et al. 1989! Diabetes 38:1326-1328). Non-specific immunostimulation caused by cytokine (Jacob et al. 1990! Proc. Natl Acad. Sci. USA 87:968-972) or adjuvant (Sadelain et al. 1990! Diabetes 39:583-589) treatments, or environmental microbes have been implicated in other protocols of IDD prevention (Like et al. 1991! Diabetes 40:259-262). An enhanced understanding of the pathogenic role of insulin autoimmunity in IDD is clearly required.
There have been reports of efforts to induce antigen-specific immunotegulation to ameliorate autoimmune diseases (Steinman, L. 1990! Mol. Biol. Med. 7:333-339). For example, various methods have been employed to induce antigen-specific suppression of experimental allergic encephalomyelitis (EAE) (PCT application WO 91/15225). Recently, several novel immunological approaches have been explored relevant to autoimmune diseases such as EAE in mice and rats and lupus nephritis in MRL/lpr mice. Many have been directed toward blocking the function of the effector CD4.sup.+ T cell which has been shown to exhibit V.sub..beta. isotype restriction in EAE. These approaches have included the use of anti-TCR antibodies (Acha-Orbea et al., supra), synthetic TCR peptides (Offner, H., G. A. Hashim, A. A. Vandenbark 1991! Science 251:430-432) and superantigen treatment (Kim, C., K. A. Siminovitch, A. Ochi 1991! J. Exp. Med. 174:1431-1437). The tolerogenic effects of intragastric antigens administration are also well described (Silverman et al. 1983! J.Immunol. 131:2651-2661; Peng et al. 1990! Clin. Exp. Immunol. 81:510-515; Michael 1989! Immune Invest. 18:1049-1054; Kitamura et al. 1987! J. Immunol. 139:3251-3259; Michalek et al. 1982! J.Immunol. 128:1992-1998; Mowat, A. M. et al. 1986! Immunol. 58:677-683; PCT applications WO 91/12816, WO 91/01333; WO 92/06704). Nagler-Anderson et al. (1986! Proc. Natl. Acad. Sci. USA 83:7443-7446) describe the oral administration of collagen-induced arthritis in a mouse model. Deficiencies in this ability have been reported in several experimental mouse models of autoimmune diseases (Gesualdo et al. 1990! J.Immunol. 145:3684-3691; Miller et al. 1984! Clin. Immunol. Immunopathol. 31:231-240).
Zhang et al. observed a beneficial response to oral insulin (Zhang et al. 1991! Proc. Natl. Acad. Sci. USA 88:10252-10256) and further demonstrated in co-transfer studies that splenocytes from insulin-fed NOD mice prevented the adoptive transfer of diabetes by splenocytes from untreated, diabetic mice to irradiated recipients.
Our current invention provides the means for the first time to prevent the disease process from becoming established in the genetically predisposed, or otherwise in progressing once initiated as identifiable by autoantibodies, through a novel treatment that is akin to vaccination to self proteins important to the pathogenic process underlying IDD.